pharmacokinetic and pharmacodynamic characterization of a novel formulation containing co formulated interferons alpha 2b and gamma in healthy male volunteers

The aim of this work was to characterize the pharmacokinetics and pharmacodynamics of a novel formulation containing a co-formulated combination of IFNs alpha-2b and gamma CIGB-128-A.. T

R E S E A R C H A R T I C L E Open Access

Pharmacokinetic and pharmacodynamic

characterization of a novel formulation

containing co-formulated interferons

alpha-2b and gamma in healthy male volunteers

Idrian García-García1, Ignacio Hernández-González2, Alina Díaz-Machado3, Carlos A González-Delgado3,

Sonia Pérez-Rodríguez3, Yanelda García-Vega1, Rosario Campos-Mojena1, Ángela D Tuero-Iglesias1,

Carmen M Valenzuela-Silva1, Alieski Cruz-Ramírez1, Alis Martín-Trujillo3, Héctor Santana-Milián4,

Pedro A López-Saura1, Iraldo Bello-Rivero1*for the CIGB-128-A Study Group

Abstract

Background: More potent antitumor activity is desired in Interferon (IFN)-treated cancer patients This could be achieved by combining IFN alpha and IFN gamma The aim of this work was to characterize the pharmacokinetics and pharmacodynamics of a novel formulation containing a co-formulated combination of IFNs alpha-2b and gamma (CIGB-128-A)

Methods: A group of nine healthy male subjects received intramuscularly 24.5 × 106IU of CIGB-128-A IFN

concentrations were evaluated for 48 h Serum neopterin, beta2-microglobulin (β2M) and 2′–5′ oligoadenylate synthetase (2′–5′ OAS), classical IFN-inducible serum markers, were measured during 192 h by enzyme

immunoassay and body temperature was used as pharmacodynamic variable as well

Results: Concerning pharmacokinetics, serum IFNs’ profiles were better fitted to a mono-compartmental model with consecutive zero order and first order absorption, one bioavailability value No interferences by simultaneous administered IFNs were observed in their typical similar systemic profiles Neopterin andβ2M time profiles showed

a delay that was efficiently linked to pharmacokinetics by means of a zero order absorption rate constant Neopterin level was nine-fold higher than initial values, 48 h post-administration, an increment not described before At this time, mean serumβ2M peaked around the double from baseline Serum concentrations of the enzyme 2′–5′ OAS was still elevated on the 8 day post-injection The formulation was well tolerated Most frequent adverse reactions were fever, headache, arthralgia and lymphopenia, mostly mild

Conclusions: The administration of co-formulated IFN alpha-2b and IFN gamma likely provides improved

pharmacodynamic properties that may be beneficial to treat several malignancies

Trial registration: Cuban Public Registry of Clinical Trials RPCEC00000118, May 24, 2011

Keywords: Interferons, Pharmacokinetics, Pharmacodynamics, Neopterin, Beta2-microglobulin, 2′–5′ oligoadenylate synthetase

* Correspondence: iraldo.bello@cigb.edu.cu

1 Clinical Research Direction, Center for Genetic Engineering and

Biotechnology, Ave 134 b/23 and 25, Cubanacán, Playa, P.O Box 6332,

Havana, Cuba

Full list of author information is available at the end of the article

© The Author(s) 2016 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made The Creative Commons Public Domain Dedication waiver

Approaches to improve efficacy, tolerance and patient’s

compliance and quality of life are permanent issues in

pharmacological therapy Some efforts have succeeded

in favoring the bioavailability of the modified drug Other

actions are based on extending the systemic half-life

through slower absorption

Interferons (IFNs), like other low molecular weight

protein drugs, have a relatively short half-life Their

conjugation to polyethylene-glycol (PEG) represented a

favorable step forward to face this drawback [1]

Never-theless, improved pharmacodynamics by the

combin-ation of two agents that have the potential to act

synergistically can be another reasonable alternative

In that sense, IFN alpha-2b and IFN gamma have

rec-ognized synergistic antiproliferative effects on several

tumor cell lines [2] based on the expression and

activa-tion of several IFN regulated genes [3] The physical

interaction between both IFN receptor complexes seems

to be the first step for triggering intracellular signals that

promote the potentiation of biological activities between

both IFNs [4, 5]

The experience of the Center for Genetic Engineering

and Biotechnology (CIGB, in Spanish) in the

develop-ment of several formulations that contain recombinant

molecules has allowed to obtain a novel formulation

(CIGB-128-A) based on the combination of IFNs

alpha-2b and gamma mixed in antiproliferative synergistic

pro-portions defined in vitro [6] The peri- and intralesional

administration of the co-formulated IFNs was safe and

effective for the treatment of elder patients with

ad-vanced, recurrent or resistant to previous treatments

basal and squamous cell skin carcinomas [7] In patients

with mycosis fungoides, a similar IFN combination did

not modify the kinetics of individual IFNs’ concentrations,

but it produced a significant increment in neopterin

serum levels, a well-known pharmacodynamic measure in

IFN studies [8]

As part of CIGB-128-A biological characterization, it

was necessary to carry out a clinical study in healthy

male subjects to integrally analyze pharmacokinetics and

pharmacodynamics, using available modeling tools This

population has a less inter-individual variability than

on-cologic patients In order to describe the kinetic behavior

of IFN-induced response, neopterin, β2-microglobulin

(β2M), 2′–5′ oligoadenylate synthetase (2′–5′ OAS) as

well as body temperature were used as pharmacodynamic

variables

Methods

A phase one clinical trial was carried out at the National

Center for Toxicology, in Havana, a certified reference

unit for this type of studies

Subjects

Inclusion criteria were: no history of chronic diseases,

no acute illness in the previous 30 days, no symptoms or signs at physical examination and laboratory tests, and

no presence of HIV and hepatitis B and C virus infection markers in serum Toxic habits, history of hypersensibil-ity to any drug, treatment with any IFN formulation in the previous 6 months or with any drug in the previous

15 days, surgical intervention in the previous 6 months and blood donations in the previous 2 months were ex-clusion criteria Subjects could withdraw from the trial voluntarily, due to occurrence of severe adverse reac-tions, or by the appearance of any exclusion criteria

IFN formulation

CIGB-128-A, Heber Biotec, Havana, a stabilized lyophi-lized powder formulation was used Each vial contains 3 and 0.5 × 106IU of human recombinant IFNs alpha-2b and gamma, respectively, both produced in E coli, tre-halose, succinic acid and human serum albumin

Study design

Each volunteer received 24.5 × 106 IU of CIGB-128-A intramuscularly, in the gluteus region This represents

21 × 106 IU of IFN alpha-2b and 3.5 × 106 IU of IFN gamma For this, the content of seven vials was diluted

in 2 mL of water for injection Detectable serum levels

of both IFNs and their surrogate markers were expected

at this dose level The product was administered early in the morning after overnight fasting Simultaneously with CIGB-128-A injection and thereafter, volunteers received oral antipyretic medication in order to mitigate flu-like symptoms produced by IFN Volunteers were regularly checked for vital signs and adverse manifestations dur-ing the study They were hospitalized durdur-ing the first

48 h, thereafter ambulatory monitoring and blood sam-pling continued until 8 days

Laboratory evaluations

For IFN alpha-2b and IFN gamma measurements, sam-ples were collected by venipuncture before injection and after 2, 3, 4, 6, 7, 8, 10, 12, 14, 16, 24, 36, and 48 h Neopterin,β2M and 2′–5′ OAS determinations in serum were extended until 192 h after injection These vari-ables were assessed before injection and after 6, 12, 24,

48, 72, 96, 120, 168, and 192 h after Some routine hematological (hemoglobin, platelet, leukocyte counts) and biochemical (hepatic enzymes, creatinine) determi-nations were evaluated for safety during the sampling period

All the pharmacological variables were measured using commercially available enzyme immunoassay (EIA) kits, IFN alpha (Bender MedSystem, Germany, LOQ: 3.2 pg/mL, CV: 7.2 %), IFN gamma (Bender MedSystem,

Germany, LOQ: 0.99 pg/mL, CV: 5.7 %), neopterin

(HENNING test, BRAHMS Diagnostica, Berlin, Germany,

LOQ: 0.5 ng/mL, CV: 6.9 %), β2M (Quantikine® IVD®,

R&D System, UK, LOQ: 0.2μg/mL, CV: 7.1 %) and 2′–5′

OAS isoform 1 (USCNK Life Science, USA, LOQ <1.5 ng/

mL, CV < 12 %) In all cases, sera were stored at−70 °C

until testing Clinical laboratory variables were tested

using advanced automated analyzers

Data analysis

Serum concentration versus time profiles of IFN

alpha-2b and IFN gamma were analyzed using MONOLIX

(Lixoft S.A.S, version 4.3.2, 2014, Paris, France) A

previ-ous evaluation showed the combined observational

mod-eling as the most appropriated A log-normal parameter

distribution was selected, except for absorbed fraction

(F), which was considered as logit-normal Seven

alter-native compartmental models were evaluated, according

with the number of compartments and the complexity

of the absorption process Table 1 describes models

considered

The objective function for model selection was the

Akaike Information Criterion (AIC) Additionally, we

considered the coefficient of correlation between

individ-ual predicted values and actindivid-ual measured concentration

Area under the curve (AUC) was calculated by means

of the trapezoidal rule from measured individual

con-centrations and from model predicted concentration

values AUC relative prediction error also contains

in-formation about model adequacy and is calculated as

follow

PEð Þ ¼% AUCp‐AUCm

Where AUCpis the area calculated from individual

pre-dicted concentrations and AUCm is the area calculated

from measured concentration values As a measure of

bias, we considered the mean relative prediction error

and its root mean square as precision assessment [9]

We used the following relation for clearance calculation

An integrated pharmacokinetics/pharmacodynamics (PK/PD) model was written using the build-in MLXTRAN code in MONOLIX, based on the best-fit PK model and a classical indirect response model with response stimula-tion [10]

dR

dt ¼ k0

in⋅ 1 þ Smax⋅Cp

SC50þ Cp

‐kout⋅R

Where R is the response, k0in is the zero order rate con-stant for the production of response, koutis the first order rate constant for response decrement and Cpis the serum concentration, Smaxis the maximum stimulatory factor at-tributed to drug and SC50is drug concentration producing

50 % of maximum stimulation

To account for differences in response between both IFNs related to pharmacokinetics, a delay equivalent to the duration of zero order absorption (Tk0) was intro-duced, manifest in the case of neopterin and β2M For 2′–5′ OAS and body temperature, initial response level was added to the model A diagram to illustrate the final adopted structural model is shown in Fig 1

Running integrated PK/PD model in MONOLIX, we left constant pharmacokinetic parameters and its stand-ard deviation (fixed and random effects) as previously estimated, allowing the system to estimate individual pharmacokinetic parameters and population and individ-ual pharmacodynamic parameters

Description of pharmacokinetic and pharmacodynamic parameters were done using Microsoft Excel 2010 (Microsoft Corp.; USA) data analysis package Categorical analysis (Wald’s test) was performed with MONOLIX Vital signs, hematological counts and blood chemistry were analyzed using paired analysis (Student’s t test or Wilcoxon’s test) depending on the normality assumption, taking into account Bonferroni adjustment for multiple

Table 1 Model description of the seven compartmental models used and estimated parameters

V Mono-compartmental with consecutive zero order and first order absorption One bioavailability value Tk0, ka, F, V, k

VII Mono-compartmental with consecutive zero order and first order absorption One bioavailability value for

each absorption component

Tk0, ka, F1, F2, V, k Tk0 duration of zero order absorption, F bioavailability associated to absorption processes, V volume of distribution of central compartment, ka rate constant of

comparisons SPSS for Windows version 15.0 was the

soft-ware used for these safety analyses

Results

Nine healthy male volunteers received the tested

formu-lation from a total of 19 subjects that were checked This

sample size was estimated using the method for 95 %

confidence interval of a mean taken into account

previ-ous pharmacodynamic results obtained in patients with

mycosis fungoides [8] Skin white color prevailed (66.7 %)

Their age ranged from 23 to 39 years-old (28 ± 5 years),

weighed 52 to 98 Kg (71 ± 16 Kg) and were 162 to 175 cm

(170 ± 5 cm) tall

Pharmacokinetic analysis

The best-fit model was number V for both IFNs Table 2

shows AIC and correlation coefficient, while Table 3

summarizes the pharmacokinetic parameters estimated

according to model V

Figures 2 and 3 depict the relation between measured

and predicted concentration as well as the visual

predic-tion checking (VPC) according to model V for IFNs

alpha-2b and gamma, respectively Raw data is plotted

together with VPC graph

Interestingly, IFN gamma also fit well to model V, but

with a shorter duration of the zero order absorption step

(p < 1×10−10, Wald’s test) and a higher volume of

distri-bution (p = 0.008, Wald’s test)

Average serum IFN profiles were qualitatively similar,

although the maximum was reached 3–4 h before for

IFN gamma IFN gamma concentrations were higher, in

agreement with the administered mass, calculated from

their respective specific activities Pre-dose serum IFN

concentration was undetectable or very low Most vol-unteers bordered or surpassed 100 pg/mL of IFN alpha and 200 pg/mL of IFN gamma

Obtained AUC48covered more than 96 % of AUC ex-trapolated to infinity Table 4 shows areas under the curve of pharmacokinetic profiles for measured and pre-dicted concentrations Figure 4 represents the correlation between actual and predicted AUC for IFN alpha-2b and IFN gamma, respectively It is noticeable that there is an excellent correspondence between actual and predicted AUC, which demonstrates the adequacy of best-fit model

Pharmacodynamic analysis

Table 5 contains AIC and R2for each measured response obtained from the application of the selected model As observed, this model fits better in the case of IFN alpha than in the case of IFN gamma according to AIC

Table 6 summarizes pharmacodynamic parameters and its variability expressed as confidence intervals Both IFNs were co-administered; therefore, the response due

to each IFN cannot be separated Nonetheless, according with our results IFN alpha-2b fits better to the selected PK/PD model Only for body temperature, a similar in-crease and dein-crease in the response is observed when each IFN is separately modeled

Figures 5 and 6 depict the VPC according to the se-lected integrated PK/PD model for IFNs alpha-2b and gamma, respectively It is evident the adequacy of the in-tegrate PK/PD model for IFN alpha-2b with neopterin and β2M, because Tk0 is almost equivalent to response initial delay, shorter in the case of IFN gamma

Baseline levels of biomarkers were within normal values specified by the EIA kits suppliers A strong neopterin

Fig 1 Schematic PK-PD structural model for IFN response

Table 2 Values of objective function and correlation between measured and predicted concentrations

AIC Akaike Information Criterion

Table 3 Mean individual pharmacokinetic parameters and confidence intervals for IFN alpha-2b and IFN gamma (Model V)

Parameter

(units)

Fig 2 Relation between measured and predicted IFN alpha-2b concentrations in serum Legend: Data correspond to IFN alpha-2b concentrations (pg/mL) measured by EIA after a single intramuscular administration of 24.5 × 10 6 IU of CIGB-128-A to the nine healthy male subjects, according

to model V a Population predicted concentration b Individual predicted concentration and (c) VPC graph

production was obtained with co-formulated IFNs An

ap-proximately nine-fold average increase was obtained 48 h

after CIGB-128-A administration (Figs 5a and 6a)

Neopterin values remained clearly upper baseline levels

between 12 and 120 h All subjects increased this marker

at least by six times compared to pre-dose values

Average β2M approximately doubled the initial value

between 24 and 48 h after injection, and then slowly

returned to baseline at the end of the sampling period (Figs 5b and 6b) Maximum levels of 2′–5′ OAS1 were achieved 6–12 h after CIGB-128-A administration; these were around 18-fold higher than baseline Twelve hours later, OAS concentration was reduced, but levels remained stable from 96 h on, being ap-proximately four-fold higher than baseline (Figs 5c and 6c)

Fig 3 Relation between measured and predicted IFN gamma concentrations in serum Legend: Data correspond to IFN gamma concentrations (pg/mL) measured by EIA after a single intramuscular administration of 24.5 × 10 6 IU of CIGB-128-A to the nine healthy male subjects, according

to model V a Population predicted concentration b Individual predicted concentration and (c) VPC graph

Table 4 Actual and predicted areas under the curve with its estimated bias and precision

95 % CI

Precision

95 % CI

Safety data

All volunteers presented at least one adverse event,

predominantly flu-like symptoms, but none of them

withdrew from the trial due to this or other causes

Fever and headache were registered in all subjects

Other manifestations such as arthralgias, lymphopenia,

tachycardia, myalgias, malaise, tremors, anorexia and

thrombocytopenia occurred in more than half of the

in-dividuals Most events were mild (85.9 %), being well

solved No severe adverse events were recorded

Despite the antipyretic treatment, the increment in body temperature was quite remarkable, reaching 39.0 °C

in eight subjects Peak temperature values were reached between 7 and 10 h after IFN administration (Figs 5d and 6d)

White cells and platelet counts were reduced below normal values in most subjects Particularly, mean changes from baseline in lymphocyte count were significant (p < 0.006) at several times, outstandingly 24 h post-injection, when the mean value was 0.7 × 109/L Other clin-ical laboratory measures were not significantly affected

Discussion

An initial zero order absorption rate, Tk0, was also ob-served by other authors after subcutaneous administration

of IFN alpha [11] As both IFNs are co-administered in a single formulation, we cannot differentiate their individual participation in the total pharmacodynamic responses In addition, we cannot explain the high levels of a pharmaco-dynamic marker in blood because of the lack of empirical

y = 0.9704x + 44.805 R² = 0.9959

1300 1500 1700 1900 2100 2300 2500 2700

AUC from measured concentrations

y = 0.9802x - 5.3381 R² = 0.999

1300 2300 3300 4300 5300 6300 7300 8300 9300

1300 2300 3300 4300 5300 6300 7300 8300 9300

AUC from measured concentrations

a

b

Fig 4 Correlation between actual and predicted AUC (pg · h/mL) for (a) IFN alpha-2b and (b) IFN gamma

Table 5 Criteria of integrated PK/PD model adequacy for each

measured response

IFN Criteria Neopterin β 2 M 2 ′–5′ OAS1 Temperature

AIC Akaike Information Criterion

data to“fill” compartments defined in complex structural

models based on mechanistic approaches This situation is

very common in clinical trials [12], like the present study

Pharmacodynamics of CIGB-128-A formulation was

characterized through three well-known IFN response

markers, mediators of their main biological actions,

trad-itionally used in this type of studies [13, 14] Neopterin

is an excellent marker of the immune-mediated

cytotox-icity and induction of apoptosis in human malignancies

[15] Beta2-microglobulin plays a key role in tumor

growth control and metastases [16] The 2′–5′OAS

en-zyme interferes in the progression of the cell cycle [17]

and produces degradation of viral RNA [18]

The delayed irruption of neopterin andβ2M responses

is commonly explained as a consequence of the complex

intracellular pathways triggered after IFN binding to its

surface receptor This concept was successfully applied

to the stimulation of the synthesis of the antiviral Mx

protein by IFN alpha-2a subcutaneous administration to

healthy volunteers [19]

Working with the obtained data, delay in neopterin andβ2M responses could be related to Tk0 Existence of zero order absorption has been suggested to occur owing

to saturated absorption just after administration [11] Dif-ferences in molecular structure, consequently in physico-chemical characteristics, can be a plausible cause for differences in absorption between IFNs alpha and gamma However, a delay was not observed for the other mea-sured responses (2′–5′ OAS, temperature) which could obey to different triggering mechanisms [20] Quantifica-tion of serum or plasma 2′–5′ OAS by EIA (instead of intracellular expression or enzymatic activity) to evaluate pharmacodynamics in humans is not easily identified in literature Body temperature was used as pharmacody-namic variable as well, although its increment consti-tutes an undesired effect IFN alpha induces fever, at least partly due to direct effects on the hypothalamic thermosensitive neurons, which involve also the opiate receptor mechanism [21]; while IFN gamma stimulates the release of pyrogenic interleukin-1 [22]

On the other hand, there are no data indicating interfer-ences in the pharmacokinetic profiles of IFNs alpha-2b and gamma when they are simultaneously administered Our findings reveal a pharmacokinetic behavior of IFN alpha-2b quite similar to that reported by other authors [11] Distribution to a wide range of tissues and organs and a very fast clearance are distinctive characteristics of IFNs [23–27]

Despite IFNs antitumor activity being well-known at present, no major advances have been achieved in the treatment of solid and hematological malignancies in the last decades An enhanced survival or a better objective response cannot be achieved by a dose increment since more severe adverse reactions could irrupt Additionally,

a more prolonged schedule reduces patient’s compliance

A sustained full IFN-receptor interaction that triggers strong antiproliferative activities is desired in the cancer treatment with this cytokine The slow systemic clear-ance of PEG-IFN alpha structures had led to less dosing, more efficacy and less toxicity in patients with chronic viral hepatitis [28] However, pegylation produces a notable reduction in IFN bioactivity [29], probably due

to a non-optimal interaction with its receptor Add-itionally, high molecular weight pegylated IFNs could have more difficulties to penetrate into the tumor microenvironment and have an effective interaction with its receptor

The combination of co-formulated IFNs alpha and gamma could improve antitumor effects of separate IFNs CIGB-128-A formulation was developed on the basis

of two essential criteria; first, the in vitro and in vivo po-tentiation of common biological activities of IFN alpha-2b and IFN gamma in certain proportions previously defined [6, 30] and second, their similar pharmacokinetics

Table 6 Mean individual pharmacodynamic parameters and

confidence intervals for IFNs alpha-2b and gamma

Parameter

(units)

Neopterin

k in (h−1) 0.027 0.024 –0.030 0.03 0.026 –0.034

k out (h−1) 0.022 0.022 –0.022 0.0247 0.0247 –0.0248

β2-microglobulin

k in (h−1) 0.141 0.133 –0.149 0.04 0.033 –0.038

k out (h−1) 0.070 0.067 –0.073 0.0194 0.0190 –0.0198

2 ′–5′ oligoadenylate synthetase

k out (h−1) 28.20 14.21 –42.18 17.7 6.2 –29.1

Body temperature

k in (h−1) 0.864 0.858 –0.870 0.851 0.848 –0.855

k out (h−1) 1.0155 1.015 –1.016 1.021 1.021 –1.021

S 0 baseline response, S max maximum stimulatory factor attributed to drug,

SC 50 : drug concentration producing 50 % of maximum stimulation, k in the zero

order rate constant for the production of response, k out the first order rate

constant for loss of response

0 20 40 60 80 100 120 140 160 180 200

0

2

4

6

8

10

12

14

16

18

20

time (h)

Data emp prctile P.I 90%

P.I 10%

0 20 40 60 80 100 120 140 160 180 200 1

1.5 2 2.5 3 3.5 4 4.5 5 5.5 6

time (h)

Data emp prctile P.I 90% P.I 10%

0 20 40 60 80 100 120 140 160 180 200

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

time (h)

Data emp prctile P.I 90%

P.I 10%

0 20 40 60 80 100 120 140 160 180 200 35

36 37 38 39 40

time (h)

Data emp prctile P.I 90% P.I 10%

a

o C

c

b

d

Fig 5 Model validation and raw data of pharmacodynamic variables for IFN alpha-2b Legend: Data correspond to the nine healthy male subjects who received 24.5 × 10 6 IU of CIGB-128-A at time 0 a Neopterin b β 2 M c 2 ′–5′ OAS1 and (d) Body temperature (first 48 h) Raw data is plotted together with VPC graph

0 20 40 60 80 100 120 140 160 180 200 35

36 37 38 39 40 41 42 43

time (h)

Data emp prctile P.I 90% P.I 10%

0 20 40 60 80 100 120 140 160 180 200

0

2000

4000

6000

8000

10000

time (h)

Data emp prctile P.I 90%

P.I 10%

0 20 40 60 80 100 120 140 160 180 200 1

2 3 4 5 6 7 8

time (h)

Data emp prctile P.I 90% P.I 10%

0 20 40 60 80 100 120 140 160 180 200

0

5

10

15

20

25

time (h)

Data emp prctile P.I 90%

P.I 10%

a

o C

c

b

d

Fig 6 Model validation and raw data of pharmacodynamic variables for IFN gamma Legend: Data correspond to the nine healthy male subjects who received 24.5 × 10 6 IU of CIGB-128-A at time 0 a Neopterin b β 2 M c 2 ′–5′ OAS1 and (d) Body temperature (first 48 h) Raw data is plotted together with VPC graph

The high increments in serum neopterin levels in

healthy subjects could support the hypothesis on which

CIGB-128-A formulation was conceived That nine-fold

neopterin increase has not been reported with any

sub-type or variant of IFN After a single intramuscular dose

of 18 × 106 IU IFN beta to healthy volunteers, serum

neopterin increments were five-fold higher than baseline

levels [13], leading to a lower Smax in a later modeling

analysis [31] Single subcutaneous doses of 27 and 36 × 106

IU of IFN alpha-2a to healthy volunteers only produced

four-fold increments in plasma neopterin concentrations

[32] The induction of neopterin by PEG-IFN alpha in

pa-tients or healthy volunteers only approximately tripled the

initial values, 48 h after injection [14, 33, 34] Pegylation

seems to produce a reduction in Smaxas occurred with Mx

protein [19] In the previous clinical trial in patients with

mycosis fungoides, intramuscular 23 × 106 IU of the

co-formulated IFNs produced neopterin increments that were

six-fold higher at the same sampling time [8]

Beta2-microglobulin increments are around 60 % after

administration of PEG-IFN alpha [14, 34], lower than

100 % found 24–48 h after CIGB-128-A injection

Al-though this last increment was reached in a similar

popula-tion after administrapopula-tion of 20 ×106 IU of IFN alpha-2b

[27], a slower return to initial levels was now observed

Six hours after a single CIGB-128-A intramuscular

injection, 2′–5 OAS1 serum levels were extensively

in-creased in healthy volunteers In IFN-treated patients,

enzymatic activity of 2′–5 OAS appears to increase

since 6 h and maintains elevated levels until 4–8 months

later during chronic treatments [35] OAS induced by

CIGB-128-A continued detectable until 192 h and the

moment to return to normality could not be predicted

Nevertheless, we need to deepen into the mechanisms

that trigger all these responses allowing us to explain the

postulated enhanced effect There are other factors

making this task difficult, mainly those related to

administered doses, like response saturation [32]

Pharmacodynamic findings were part of the overall

in-formation that justified approval of further clinical trials

with CIGB-128-A formulation In these trials more spaced

dosing schedules are evaluated in patients with advanced

or metastatic solid tumors without other therapeutic

options These are ongoing trials and preliminary results

are encouraging

The formulation was well tolerated As expected, flu-like

symptoms and hematological count reductions were the

most common adverse events generated after

administra-tion Bone marrow depression produced after only one IFN

dose can be explained by increase in cortisol levels [36]

Conclusions

In this work PK/PD of CIGB-128-A formulation,

contain-ing IFNs alpha 2b and gamma co-formulated in synergistic

proportions, was characterized The PK best-fit model was number V for both IFNs and average serum IFN profiles were qualitatively similar Compared with literature data CIGB-128-A promotes strong neopterin and 2–5 OAS production and extends upper baseline levels until 98 and

120 h, respectively A unique dose of more than 20 MIU

of CIGB-128A was safe with no severe event reported These properties indicate that this formulation is a good candidate to treat some oncologic diseases, without phar-macokinetic interferences or additional toxicity

Abbreviations

2 ′–5′ OAS: 2 ′–5′ oligoadenylate synthetase; AIC: Akaike Information Criterion; AUC: Area under the curve; CI: Confidence intervals; Cl: Clearance;

EIA: Enzyme immunoassay; IFN: Interferon; PD: Pharmacodynamics; PEG: Polyethylene –glycol; PK: Pharmacokinetics; β 2 M: Beta2-microglobulin Acknowledgments

The authors wish to thank Iván Campa, Grettel Melo, Ketty Cruz and María A Delgado for their participation, Cimara Bermúdez, Greisy Pérez, Yunia Delgado, Elizeth García and Yamilet Vázquez for their assistance in the clinical work, Edgar Casanova, PhD, for manuscript review and suggestions, and especially the nine young men who served as volunteers The authors received CIGB-128-A formulation free from Heber Biotec, Havana.

The other members of the CIGB-128-A Study Group are: Ivonne Rivero-Vázquez, Laura Barrero-Viera, Maylén Álvarez-Delgado, Maura Tamayo-Rodríguez, Marlene David-Baldo, Lourdes Olivera-Ruano, Gricel González-Gamiz (National Center for Toxicology), Majel Cervantes-Llano, Reinier Hernández-Rodríguez, Leovaldo Álvarez-Falcón (Center for Genetic Engineering and Biotechnology), Ivón Howland-Álvarez, Yolanda Cruz-Gómez (Center for Medical-Surgical Research, Havana, Cuba).

Funding The study was financed by Heber Biotec, Havana, Cuba (product, reagents) The Ministry of Public Health of Cuba supported the clinical trial (hospital facilities and general medical care of the volunteers as in-patients) Availability of data and materials

The data sets supporting the results of this article are included within the article.

Authors ’ contributions IGG designed and coordinated the study, performed the immunoassays, analyzed the results, and wrote the manuscript draft IHG performed PK/PD modeling ADM, CAGD and SPR took care of subject recruitment, management, clinical examinations, and follow-up YGV and PALS participated in the design and revised the manuscript RCM and ACR carried out EIA determinations ADTI and CMVS contributed as data processor and statisticians AMT processed blood samples and coordinated clinical laboratory evaluations HSM developed the formulation IBR conceived the study and took part in the design, coordination, results analysis and manuscript writing All authors read and approved the final manuscript.

Competing interests Authors IGG, YGV, RCM, ADTI, CMVS, ACR, HSM, PALS and IBR are employees

of the Center for Genetic Engineering and Biotechnology (CIGB), Havana network, where IFN alpha-2b and IFN gamma are produced and the novel formulation (CIGB-128-A) was developed The rest of the authors have no competing interests at all.

Consent for publication Not applicable.

Ethics approval and consent to participate The clinical protocol was approved by the institutional ethics committee of the National Center for Toxicology in Havana All procedures performed in healthy volunteers were in accordance with the ethical standards of this committee and with the 1964 Helsinki declaration and its later amendments.